1. Field of the invention
This invention relates to positively charged non-natural amino acids, methods of making thereof, and utilization thereof in peptides. In one embodiment, the invention relates to non-natural amino acids that closely replicate the natural amino acids lysine and arginine.
2. Background
Synthetic studies have been directed toward understanding the influence that only some non-natural amino acids have on the structural and biological activity of peptides.
U.S. Pat. No. 3,178,472 to Hellerbach et al. discloses a method for the conversion of amino carboxylic acids to their N-monomethyl derivatives. Naturally occurring amino acids such as alanine, phenylalanine, serine, cysteine, cystine, tyrosine, tryptophan, histidine, methionine, valine, norvaline, leucine, isoleucine, arginine, ornithine, lysine, aspartic acid, glutaminic acid, threonine, xcex1,xcex3-diaminobutyric acid and the like are suitable starting materials. When the starting material aminocarboxylic acid contains two amino groups such as lysine, ornithine or xcex1,xcex3-diaminobutyric acid, then it is possible to generate a product in which one or both amino groups are methylated.
Moore et al. (Can. J. Biochem. 1978, 56, 315) discloses the effect of the basic amino acid side chain length and the penultimate residue on the hydrolysis of benzoyldipeptides by carboxylicpeptidase B1 (CPB). Non-natural amino acids including homolysine and homoarginine were incorporated into small peptide chains, and the kinetic prameters were determined for the CPB catalyzed hydrolysis of the peptide.
Lindeberg et al. (Int. J. Peptide Protein Res. 1977, 10, 240) discloses the synthesis of 1-deamino-4-L-valine-8-DL-homolysine-vasopressin and protected 1-deamino-4-L-valine-8-D-lysine-vassopressin in which with non-natural amino acids were incorporated. The addition of a methylene group to lysine and arginine to generate the non-natural amino acids homolysine and homoarginine, respectively. The study revealed that peptides with homolysine and homoarginine reduced the antidiuretic activity of the peptides.
Hilpert et al. (J. Med. Chem. 1994, 37, 3889) discloses screening of small basic molecules for binding in the recognition pocket of thrombin led to the discovery of the arginine mimetic (aminoiminomethyl)piperidine as a weak thrombin inhibitor. A number of derivatives of the arginine mimetic were prepared, and their ability to inhibit thrombin was assayed. The X-ray crystal structure analysis of thrombin as well as modeling studies of the arginine mimetic were conducted in order to rationalize the observed affinity between the unnatural amino acid and thrombin.
Nestor et al. (J. Med. Chem. 1988, 31, 65) discloses the synthesis of a new series of unnatural amino acids and their incorporation into antagonistic analogues of lutenizing hormone-releasing hormone (LH-RH). In particular, non-natural amino acids of arginine exhibited high acute potency and very prolonged duration of action. Biological and clinical pharmacology studies revealed that these LH-RH antagonists cause mast cell degranulation, and were removed from consideration as candidates for fall commercial development.
There is a need in the art for non-natural amino acids and for peptides incorporating such acids to achieve superior effects, such as, for example, improved diagnostic or disease fighting activity. None of the above references discloses the non-natural amino acids of the present invention or the advantageous properties thereof. The present invention accordingly describes positively charged non-natural amino acids, methods of making thereof, and utilization thereof in peptides.
Additional advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The present invention provides non-natural amino acids of lysine and arginine and their derivatives (i.e., the compounds or compositions formed from performing a specified reaction) and methods for their use.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates a non-natural amino acid compound of the formula I: 
wherein
n is an integer of from 2 to 4,
R1, R2, R3 and R13 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex1 and Cxcex2 are carbon atoms and the stereochemistry at Cxcex1 and Cxcex2 is, independently, either R or S;
or the ester or salt thereof, wherein
when R1, R2, R3 and R13 are all hydrogen, then n is not 2 or 3,
when n is 2 or 3, R1 and R13 are hydrogen, and R2 and R3 are independently hydrogen or methyl with at least one of R2 and R3 being methyl, then the stereochemistry at Cxcex2 is not S, and
when n is 4, and R1, R2, R3 and R13 are all hydrogen, then the stereochemistry at Cxcex2 is not R.
The invention further relates to a non-natural amino acid compound of the formula II: 
wherein
n is an integer of from 2 to 4;
when dashed line a is not present, X and Y are independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5;
when dashed line a is present, Xxe2x80x94Y is (CH2)z, wherein z is an integer of from 2 to 4;
R4 and R5 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at Cxcex2 is either R or S;
or the ester or salt thereof, wherein
when n is 3, dashed line a is not present, R4, X and Y are all hydrogen, and R5 is methyl, then Cxcex2 is not S,
when n is 3, dashed line a is not present, X and R5 are hydrogen, and Y and R4 are methyl, then the stereochemistry at Cxcex2 is not R,
when dashed line a is not present, and R4, R5, X and Y are all hydrogen, then n is not 3,
when n is 4, dashed line a is not present, X and R5 are hydrogen, and Y and R4 are the same lower branched or straight chain alkyl, then Cxcex2 is not R, and
when n is 4, dashed line a is not present, and R4, R5, X and Y are all hydrogen, then the stereochemistry at Cxcex2 is not R.
In yet another embodiment, the present invention relates to a non-natural amino acid compound of the formula III: 
wherein
n is an integer of from 2 to 4;
Xxe2x80x94Y is (CH2)z, wherein z is an integer of from 2 to 4;
R6, R7 and R8 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at Cxcex2 is either R or S;
or the ester or salt thereof.
The invention further relates to a non-natural amino acid compound of the formula IV: 
wherein
n is an integer of from 2 to 4;
R9, R10, R11, and R12 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at Cxcex2 is either R or S; or the ester or salt thereof.
In yet another embodiment, the invention relates to a peptide comprising the non-natural amino acid compound of formula I, II, III or IV.
In a further embodiment, the present invention provides a method for screening a peptide for an activity, comprising the steps of:
a) measuring an activity of a peptide having a selected amino acid sequence and comprising a natural amino acid;
b) measuring the same activity of a peptide having the same amino acid sequence but substituted independently in place of at least one natural amino acid, is a non-natural amino acid having the formula I, II, III or IV; and
c) comparing the measured activity of the peptides from steps a) and b) to determine whether the peptide of step b) has the activity.
In yet another embodiment, the present invention provides a method of treating or preventing in a subject a disease treated or prevented by the administration of a peptide, comprising administering to the subject a peptide having, substituted for a natural amino acid, at least one non-natural amino acid having the following formula I, II, III or IV.
The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein.
Before the present compounds, compositions and methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9canxe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise.
Throughout this application, where publications are referenced, the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Variables, such as R1-R13, n, z, X, Y, Cxcex1 and Cxcex2, throughout the application are the same variables as previously defined unless stated to the contrary.
In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:
The term xe2x80x9calkylxe2x80x9d as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Preferred alkyl groups herein contain from 1 to 5 carbon atoms.
The term xe2x80x9calkenylxe2x80x9d as used herein refers to a hydrocarbon group of 2 to 24 carbon atoms, with preferred groups within this class contain 2 to 5 carbon atoms, and structural formula containing a carbon-carbon double bond.
The term xe2x80x9calkynylxe2x80x9d as used herein refers to a hydrocarbon group of 2 to 24 carbon atom, with preferred groups within this class contain 2 to 5 carbon atoms, and a structural formula containing a carbon-carbon triple bond.
As used herein, especially in reference to alkyl, alkenyl and alkynyl, unless defined otherwise, the term xe2x80x9clowerxe2x80x9d refers to a moiety having from 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms.
The term xe2x80x9calkylating agentxe2x80x9d as provided herein is a compound with the structural formula RX, where R is an alkyl, alkenyl or alkynyl group as previously described, and X, which is preferably a halide such as chloride, bromide or iodide.
As used herein, the term xe2x80x9cnon-natural amino acidxe2x80x9d refers to an organic compound that has a structure similar to a natural amino acid but has been modified structurally to mimic the structure and reactivity of a natural amino acid.
As used herein, the term xe2x80x9cpeptidexe2x80x9d refers to a class of compounds composed of amino acids chemically bound together with amide linkages (CONH). Peptide as used herein includes oligomers of amino acids and small and large peptides, including polypeptides.
As used herein, the term xe2x80x9cactivityxe2x80x9d refers to a biological activity.
As used herein, the term xe2x80x9cpharmacological activityxe2x80x9d refers to the inherent physical properties of a peptide or polypeptide. These properties include but are not limited to half-life, solubility, and stability and other pharmacokinetic properties.
The structures of the non-natural amino acids of formula I and the non-natural amino acids of formula II, III and IV are similar to and closely replicate those of the naturally occurring amino acids lysine and arginine, respectively. In preferred embodiments, the compounds of the invention differ from the natural amino acids lysine and arginine, due, inter alia, a longer or shorter methylene bridge between the (i) amino/carboxyl terminus, which forms the bond between other amino acids in a peptide and (ii) the opposite functional terminus of the amino acid, preferably, the extended bridge of the invention compared to the natural amino acid bridge is one carbon length longer or shorter (i.e., the homo- or des-forms). In other preferred embodiments, the compounds of the invention have, inter alia, longer, shorter, or equivalent methylene bridge lengths and have substitutions at various moieties, form different moieties, or link moieties to form ring structures, compared to the comparable natural amino acid.
The design basis for all of the non-natural amino acids is the ability of positively-charged side chains to form electrostatic interactions (ion pairs or salt bridges) with negatively charged amino acids that can be influenced by the local environment (i.e. the structure of the amino acid.) In particular, placing an alkyl group in the vicinity of the charge destabilizes solvation of the ion and favors formation of an ion pair. Thus, the charged non-natural amino acids, when substituted in biologically active molecules for for positively charged natural amino acids arginine or lysine, can make the molecule bind better to its target if the particular residue is involved in binding. Since biological activity typically correlates directly with binding strength, better biologically active molecules can be created.
The invention, in one aspect, relates to a non-natural amino acid compound of the formula I: 
wherein
n is an integer of from 2 to 4;
R1, R2, R3 and R13 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex1 and Cxcex2 are carbon atoms and the stereochemistry at Cxcex1 and C Cxcex2 is, independently, either R or S;
or the ester or salt thereof wherein
when R1, R2, R3 and R13 are all hydrogen, then n is not 2 or 3,
when n is 2 or 3, R1 and R13 are hydrogen, and R2 and R3 are independently hydrogen or methyl with at least one of R2 and R3 being methyl, then the stereochemistry at Cxcex2 is not S, and
when n is 4, and R1, R2, R3 and R13 are all hydrogen, then the stereochemistry at Cxcex2 is not R.
In one embodiment, R1, R2, R3 and R13 are independently, hydrogen or lower branched or straight chain alkyl of C1-C5, preferably hydrogen or methyl. In another embodiment, n is 4. In one embodiment, the compound is:
a) n is 4, R1, R3 and R13 are hydrogen, R2 is methyl, the compound of formula I is an acid, and the stereochemistry at Cxcex2 is R;
b) n is 4, R1, R3 and R13 are hydrogen, R2 is methyl, the compound of formula I is an acid, and the stereochemistry at Cxcex2 is S;
c) n is 4, R1 and R2 are methyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is R, and the stereochemistry at Cxcex2 is R;
d) n is 4, R1 and R2 are methyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is S, and the stereochemistry at Cxcex2 is R;
e) n is 4, R1 and R2 are methyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is R, and the stereochemistry at Cxcex2 is S;
f) n is 4, R1 and R2 are methyl, R3 and R13 is hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is S, and the stereochemistry at Cxcex2 is S;
g) n is 4, R1 is methyl, R2, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is R, and the stereochemistry at Cxcex2 is R;
h) n is 4, R1 is methyl, R2, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is S, and the stereochemistry at Cxcex2 is R;
i) n is 4, R1 is methyl, R2, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is R, and the stereochemistry at Cxcex2 is S;
j) n is 4, R1 is methyl, R2, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is S, and the stereochemistry at Cxcex2 is S;
k) n is 4, R1, R2, R3 and R13 are hydrogen, the compound of formula I is an acid, and the stereochemistry at Cxcex2 is S;
l) n is 3, R1 and R2 are methyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is R, and the stereochemistry at Cxcex2 is R;
m) n is 3, R1 and R2 are ethyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is S, and the stereochemistry at Cxcex2 is R;
n) n is 3, R1 and R2 are propyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is R, and the stereochemistry at Cxcex2 is S;
o) n is 3, R1 and R2 are butyl, R3 and R13 is hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is S, and the stereochemistry at Cxcex2 is S;
p) n is 2, R1 and R2 are methyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is R, and the stereochemistry at Cxcex2 is R;
q) n is 2, R1 and R2 are ethyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is S, and the stereochemistry at Cxcex2 is R;
r) n is 2, R1 and R2 are propyl, R3 and R13 are hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is R, and the stereochemistry at Cxcex2 is S;
s) n is 2, R1 and R2 are butyl, R3 and R13 is hydrogen, the compound of formula I is an acid, the stereochemistry at Cxcex1 is S, and the stereochemistry at Cxcex2 is S;
or the ester or salt thereof.
Preferably, the compound is one of the species a-k above.
The invention further relates to a non-natural amino acid compound of the formula II: 
wherein
n is an integer of from 2 to 4;
when dashed line a is not present, X and Y are independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5;
when dashed line a is present, Xxe2x80x94Y is (CH2)z, wherein z is an integer of from 2 to 4;
R4 and R5 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at Cxcex2 is either R or S;
or the ester or salt thereof, wherein
when n is 3, dashed line a is not present, R4, X and Y are all hydrogen, and R5 is methyl, then Cxcex2 is not S,
when n is 3, dashed line a is not present, X and R5 are hydrogen, and Y and R4 are methyl, then the stereochemistry at Cxcex1 is not R,
when dashed line a is not present, and R4, R5, X and Y are all hydrogen, then n is not 3,
when n is 4, dashed line a is not present, X and R5 are hydrogen, and Y and R4 are the same lower branched or straight chain alkyl, then Cxcex2 is not R, and
when n is 4, dashed line a is not present, and R4, R5, X and Y are all hydrogen, then the stereochemistry at Cxcex2 is not R.
In one embodiment, when n is 3, dashed line a is not present, X and R5 are hydrogen, and Y and R4 are the same lower branched or straight chain alkyl, then Cxcex2 is not R. In a another embodiment, R4 and R5 are, independently, hydrogen or methyl. In a preferred embodiment, dashed line a is not present, X is hydrogen or lower branched or straight chain alkyl of C1-C5, preferably methyl or ethyl, and Y is hydrogen or lower branched or straight chain alkyl of C1-C5, preferably methyl, or dashed line a is present and z is 2, preferably, n is 3. In one embodiment, the compound is:
a) n is 3, dashed line a is not present, the compound of formula II is an acid, R4 and R5 are hydrogen, X is hydrogen, Y is methyl, and the stereochemistry at Cxcex2 is R;
b) n is 3, dashed line a is not present, the compound of formula II is an acid, R4 and R5 are hydrogen, X is hydrogen, Y is methyl, and the stereochemistry at Cxcex2 is S;
c) n is 3, dashed line a is not present, the compound of formula II is an acid, R4 is methyl, R5 is hydrogen, X is hydrogen, Y is methyl, and the stereochemistry at Cxcex2 is S;
d) n is 3, dashed line a is present, the compound of formula II is an acid, z is 2, R4 and R5 are hydrogen, and the stereochemistry at Cxcex2 is R;
e) n is 3, dashed line a is present, the compound of formula II is an acid, z is 2, R4 and R5 are hydrogen, and the stereochemistry at Cxcex2 is S;
f) n is 3, dashed line a is present, the compound of formula II is an acid, z is 2, R4 is methyl, R5 is hydrogen, and the stereochemistry at Cxcex2 is R;
g) n is 3, dashed line a is present, the compound of formula II is an acid, z is 2, R4 is methyl, R5 is hydrogen, and the stereochemistry at Cxcex2 is S;
h) n is 3, dashed line a is not present, the compound of formula II is an acid, R4 and R5 are hydrogen, X is methyl, Y is hydrogen, and the stereochemistry at Cxcex2 is R;
i) n is 3, dashed line a is not present, the compound of formula II is an acid, R4 and R5 are hydrogen, X is methyl, Y is hydrogen, and the stereochemistry at Cxcex2 is S;
j) n is 3, dashed line a is not present, the compound of formula II is an acid, R4 and R5 are hydrogen, X is ethyl, Y is hydrogen, and the stereochemistry at Cxcex2 is R;
k) n is 3, dashed line a is not present, the compound of formula II is an acid, R4 and R5 are hydrogen, X is ethyl, Y is hydrogen, and the stereochemistry at Cxcex2 is S;
l) n is 2, dashed line a is not present, the compound of formula II is an acid, R4 and R5 are hydrogen, X is hydrogen, Y is methyl, and the stereochemistry at Cxcex2 is R;
m) n is 2, dashed line a is not present, the compound of formula II is an acid, R4 and R5 are hydrogen, X is hydrogen, Y is ethyl, and the stereochemistry at Cxcex2 is S;
n) n is 2, dashed line a is not present, the compound of formula II is an acid, R4 is methyl, R5 is hydrogen, X is hydrogen, Y is propyl, and the stereochemistry at Cxcex2 is S;
o) n is 4, dashed line a is present, the compound of formula II is an acid, z is 2, R4 and R5 are hydrogen, and the stereochemistry at Cxcex2 is R;
p) n is 3, dashed line a is present, the compound of formula II is an acid, z is 2, R4 and R5 are methyl, and the stereochemistry at Cxcex2 is S;
q) n is 2, dashed line a is present, the compound of formula II is an acid, z is 3, R4 is methyl, R5 is hydrogen, and the stereochemistry at Cxcex2 is R;
or the ester or salt thereof.
Preferably, the compound is one of the species a-k above.
In yet another embodiment, the present invention relates to a non-natural amino acid compound of the formula III: 
wherein
n is an integer of from 2 to 4;
Xxe2x80x94Y is (CH2)z, wherein z is an integer of from 2 to 4;
R6, R7 and R8 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl Of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at Cxcex2 is either R or S;
or the ester or salt thereof
In one embodiment, R6, R7 and R8 are independently, hydrogen or lower alkyl or straight chain alkyl of C1-C5, preferably hydrogen or methyl, even more preferably all are hydrogen. In another embodiment, z is 2 or 3, preferably 3. In a preferred embodiment, n is 3. In one embodiment, the compound is
a) n is 3, z is 2, R6, R7 and R8 are hydrogen, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is R;
b) n is 3, z is 2, R6, R7 and R8 are hydrogen, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is S;
c) n is 3, z is 3, R6, R7 and R8 are hydrogen, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is R;
d) n is 3, z is 3, R6, R7 and R8 are hydrogen, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is S;
e) n is 2, z is 2, R6, R7 and R8 are hydrogen, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is R;
f) n is 4, z is 2, R6, R7 and R8 are hydrogen, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is S;
g) n is 2, z is 3, R6, R7 and R8 are hydrogen, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is R;
h) n is 4, z is 3, R6, R7 and R8 are hydrogen, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is S;
i) n is 2, z is 2, R6, R7 and R8 are methyl, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is R;
j) n is 4, z is 2, R6, R7 and R8 are methyl, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is S;
k) n is 2, z is 3, R6, R7 and R8 are methyl, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is R;
l) n is 4, z is 3, R6, R7 and R8 are methyl, the compound of formula III is an acid, and the stereochemistry at Cxcex2 is S;
or the ester or salt thereof.
Preferably, the compound is one of the species a-d above.
The invention further relates to a non-natural amino acid compound of the formula IV: 
wherein
n is an integer of from 2 to 4;
R9, R10, R11 and R12 are, independently, hydrogen or lower branched or straight chain alkyl, alkenyl or alkynyl of C1-C5; and
Cxcex2 is a carbon atom and the stereochemistry at C62  is either R or S;
or the ester or salt thereof.
In one embodiment, R9, R10, R11 and R12 are, independently,hydrogen or lower straight or branched chain alkyl of C1-C5, preferably hydrogen, methyl or ethyl. In another embodiment, R10 is methyl. In a preferred embodiment, R9 is hydrogen, R10 is methyl, R12 is hydrogen, and n is 3. In one embodiment, the compound is:
a) n is 3, R9, R11, and R12 are hydrogen, R10 is methyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is R;
b) n is 3, R9, R11, and R12 are hydrogen, R10 is methyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is S;
c) n is 3, R9 and R12 are hydrogen, R10 and R11, are methyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is R;
d) n is 3, R9 and R12 are hydrogen, R10 and R11 are methyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is S;
e) n is 3, R9 and R12 are hydrogen, R10 is methyl, R11 is ethyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is R;
f) n is 3, R9 and R12 are hydrogen, R10 is methyl, R11 is ethyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is S;
g) n is 2, R9, R11, and R12 are hydrogen, R10 is methyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is R;
h) n is 2, R9, R11, and R12 are hydrogen, R10 is methyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is S;
i) n is 2, R9 and R12 are hydrogen, R10 and R11 are methyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is R;
j) n is 4, R9 and R12 are hydrogen, R10 and R11 are methyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is S;
k) n is 4, R9 and R12 are hydrogen, R10 is methyl, R11 is ethyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is R;
l) n is 4, R9 and R12 are hydrogen, R10 is methyl, R11 is ethyl, the compound of formula IV is an acid, and the stereochemistry at Cxcex2 is S;
or the ester or salt therof.
Preferably, the compound is one of the species a-f above.
Each of the compounds can be prepared as the acid, salt or ester. In water, the non-natural amino acids of the present invention will be charged; however; in cell membranes and other non-polar regions of the cell, the non-natural amino acids may not be charged. In one embodiment, the ester group of the non-natural amino acids of the present invention is methyl, ethyl, t-BOC or FMOC. In another embodiment, the counter-ion for the salts of the non-natural amino acids is sodium, potassium, ammonium, chloride or bromide.
Particular non-natural amino acid compounds of the invention represented by formula I can be produced according to Scheme A: 
The cyclic lactam compound V (n is 2 to 4) is commercially available from Aldrich Chemical Company. Alkylation of the amide group of the cyclic lactam V with an alkylating agent R2X and a base generates the N-alkyl compound VI. In one embodiment, n is from 2 to 4. In a preferred embodiment, the alkylating agent R2X is methyl iodide and the base is sodium hydride. Treatment of V or VI with NaOH, generates the ring-open compound VII. Protection of the amino group can be accomplished by the addition of a base and a protecting group to a solution of VII in water. In a preferred embodiment, the base is sodium carbonate and the protecting group is di-tert-butyl dicarbonate. In another embodiment, treatment of VII with a base, preferably NaOH, NaH or LDA, and an alkylating agent R13X, preferably X is iodide generates compound VIII. Compound VIII can be initially converted to an anhydride, preferably to the tert-butyl anhydride, followed by the addition of the conjugate base of a chiral auxiliary to generate IX. In a preferred embodiment, the chiral auxiliary is S-4-benzyl-2-oxazolidinone; however, the other enantiomer of the chiral auxiliary can be used as well. The addition of a base, preferably potassium hexamethyldisilazide, to IX generates an enolate, which can be quenched with an azide agent, preferably trisyl azide. Separation of the diastereoisomers of X can be accomplished by HPLC using hexane/ethyl acetate, preferably in a 1:1 ratio, as eluent. Cleavage of the chiral auxiliary to generate the acid compound XI requires a basic, oxidizing reaction media. In one embodiment, LiOH and hydrogen peroxide can be used. Hydrogenolysis of XI in the presence of a catalyst, preferably Pdxe2x80x94C, converts the azide group to the primary amine. The amino group can be then deprotonated with a base, preferably triethylamine, and can be treated with an alkylating agent R3X, preferably X is iodide, to generate compound XII, which is a non-natural amino acid compound represented by formula I.
Particular non-natural amino acid compounds of the invention represented by formula I can be produced according to Scheme B: 
The addition of hydroxide, preferably NaOH, to a solution to XIII can produce the ring-open complex XIV. In the case of XIII, when n is 2 or 3, then these compounds are commercially available (Aldrich Chemical Company). When n is 1, then compound XIII can be prepared by deprotonating cyclobutanone with a base to generate the enolate followed by quenching with an acyl halide [R1(O)X], where X is chloride, bromide or iodide. The addition of an amine salt to XIV initially generates an imineacid complex. In one embodiment, the amine salt is methylamine hydrochloride. In another embodiment, the addition of liquid NH3 can produce the imineacid. Reduction of the imineacid with a reducing agent, preferably sodium cyanoborohydride, can generate the amino complex XV. Protection of the amino group can be accomplished by the addition of a base and a protecting group to XV. In one embodiment, the base is sodium carbonate and the protecting group is di-tert-butyl dicarbonate. In another embodiment, XV can be treated with a base, preferably NaOH, NaH or LDA, and an alkylating agent R13X, preferably X is iodide, to generate compound XVI. Compound XVI can be initially converted to an ester, preferably to the tert-butyl ester, followed by the addition of the conjugate base of a chiral auxiliary to generate XVII. In a preferred embodiment, the chiral auxiliary is S-4-benzyl-2-oxazolidinone; however, the other enantiomer of the chiral auxiliary can be used as well. The addition of a base, preferably potassium hexamethyldisilazide, to XVII generates an enolate, which can be quenched with an azide agent, preferably trisyl azide (Compound XVIII). Cleavage of the chiral auxiliary to generate the acid compound XIX requires a basic, oxidizing reaction media. In one embodiment, LiOH and hydrogen peroxide can be used. Hydrogenolysis of XIX in the presence of a catalyst, preferably Pdxe2x80x94C, converts the azide group to the primary amine. The amino group can be deprotonated with a base, preferably sodium bicarbonate, and treated with a protecting agent. In one embodiment, the protecting agent is N-(9H-fluoren-2-ylmethoxy-carbonyloxy)succinimide. In another embodiment, an alkylating agent R3X, preferably X is iodide, can be added to generate a racemic mixture comprising of compounds XX and XXI. The diasteroisomers can be separated by HPLC with hexane/ethyl acetate as the eluent. When an amino group is protected with a N-(9H-fluoren-2-ylmethoxy-carbonyloxy) group (R3=FMOC), it is readily cleaved off when treated with an amine, preferably piperidine. Compounds XX and XXI are non-natural amino acid compounds represented by the formula I.
Particular non-natural amino acid compounds of the invention represented by formula II can be produced according to Scheme C: 
An alkylating agent was added to a solution of thiourea XXII. The acyclic thiourea complex XXII is commercially avalibale. In one embodiment, the alkylating agent is methyl iodide, the thiourea is N-ethyl thiourea, and the solvent is acetone. After refluxing, an organic solvent, preferably hexane, was added, and the solution was cooled to 0xc2x0 C. White crystals of XXIII were isolated and dried under reduced pressure. Compounds XXIII and XXIV were dissolved in NaOH and stirred at room temperature. In one embodiment, the concentration of NaOH is 2 N, and the solution was stirred for nine days at room temperature. The solution was brought to neutral pH by the addition of an acid, preferably concentrated HCl. The solution can be chromatographed with an eluent, preferably ammonium hydroxide, and even more preferably 1 M ammonium hydroxide. In one embodiment, the column can be a strongly-acidic cation exchange resin. Removal of solvent affords compound XXV, a non-natural amino acid compound having the formula II as the ammonium salts.
Particular non-natural amino acid compounds of the invention represented by formula III can be produced according to Scheme D: 
An alkylating agent was added to a solution of the cyclic thiourea XXVI. The cyclic thiourea complex XXVI is commercially available. In one embodiment, the alkylating agent is methyl iodide. In a preferred embodiment, the cyclic thiourea is a five- or six-member ring, and the solvent is acetone. After refluxing, an organic solvent, preferably hexane, was added, and the solution was cooled to 0xc2x0 C. White crystals of XXVII were isolated and dried under reduced pressure. Compounds XXVII and XXVIII were dissolved in NaOH and stirred at room temperature. In one embodiment, the concentration of NaOH is 1 N. In another embodiment, the solution was refluxed for 4 hours then cooled to room temperature. The solution pH can be lowered by the addition of an acid. In one embodiment, the acid is HCl and the pH is 4. The solution can be chromatographed with an eluent, preferably ammonium hydroxide, and even more preferably 1.5 N ammonium hydroxide. In one embodiment, the column is a strongly-acidic cation exchange resin. Removal of solvent affords compound XXIX, a non-natural amino acid compound having the formula III.
Particular non-natural amino acid compounds of the invention represented by formula IV can be produced according to Scheme E: 
Protection of the xcex1-amino group of an amino acid generates compound XXX. In one embodiment, the protecting group is BOC. The addition of an alkylating agent R11X in the presence of a base produces the N-alkyl compound XXXI. In a preferred embodiment, the alkylating agent is methyl iodide and the base is sodium bicarbonate. The acetimidate XXXII is added to a solution of XXXI. In one embodiment, methyl acetimidate is preferred. In another embodiment, the solvent is MeOH, and the solution was stirred at an elevated temperature, preferably 70xc2x0 C., for 4 hours. Solvent was removed under reduced pressure and the residue can be subjected to chromatography. In one embodiment, silica gel chromatography is preferred in order to isolate compound XXXIII. Deprotection of the a-amino group of XXXIII can be accomplished by the addition of an acid. In a preferred embodiment, the protecting group is BOC and the acid is trifluoroacetic acid. Removal of the protecting group generates compound XXXIV, a non-natural amino acid compound having the formula IV.
All peptides were synthesized by the Merrifield solid phase method, which is an established method for preparing peptides to those skilled in the art. All compounds having the formulas I-IV can be protected at the xcex1-amino group with standard protecting groups. In a preferred embodiment, the protecting groups are BOC and FMOC. The non-natural amino acids having the formulas I-IV can be substituted in any desired peptide, wherein the substitution is site-specific.
Screening of Non-Natural Amino Acid-containing Peptides
The invention provides a method for screening a peptide for an activity, comprising the steps of: a) measuring an activity of a peptide having a selected amino acid sequence and comprising a natural amino acid; b) measuring the same activity of a peptide having the same amino acid sequence but substituted independently in place of at least one natural amino acid, is a non-natural amino acid having the formula I, II, III or IV described above; and c) comparing the measured activity of the peptides from steps a) and b) to determine whether the peptide of step b) has the activity.
The activities for which the present invention screens can include any activity associated with a biologically active peptide or peptidomimetic. The following is a partial list of the many activities that can be determined in the present screening method:
1. Receptor agonist/antagonist activity:
A compendia of examples of specific screens for measuring these activities can be found in: xe2x80x9cThe RBI Handbook of Receptor Classification and Signal Transductionxe2x80x9d K. J. Watling, J. W. Kebebian, J. L. Neumeyer, eds. Research Biochemicals International, Natick, Mass., 1995, and references therein. Methods of analysis can be found in: T. Kenakin xe2x80x9cPharmacologic Analysis of Drug-Receptor Interactionsxe2x80x9d 2nd Ed. Raven Press, New York, 1993, and references therein.
2. Enzyme inhibition:
A compendia of examples of specific screens for measuring these activities can be found in: H. Zollner xe2x80x9cHandbook of Enzyme Inhibitorsxe2x80x9d, 2nd Ed. VCH Weinheim, FRG, 1989, and references therein.
3. Central nervous system, autonomic nervous system (cardiovascular and gastrointestinal tract), antihistaminic, anti-inflammatory, anaesthetic, cytotoxic, and antifertility activities:
A compendia of examples of specific screens for measuring these activities can be found in: E. B. Thompson, xe2x80x9cDrug Bioscreening: Drug Evaluation Techniques in Pharmacologyxe2x80x9d, VCH Publishers, New York, 1990, and references therein.
4. Anticancer activities:
A compendia of examples of specific screens for measuring these activities can be found in: I. J. Fidler and R. J. White xe2x80x9cDesign of Models for Testing Cancer Therapeutic Agentsxe2x80x9d, Van Nostrand Reinhold Company, New York, 1982, and references therein.
5. Antibiotic and antiviral (especially anti-HIV) activities:
A compendia of examples of specific screens for measuring these activities can be found in: xe2x80x9cAntibiotics in Laboratory Medicinexe2x80x9d, 3rd Ed., V. Lorian, ed. Williams and Wilkens, Baltimore, 1991, and references therein. A compendia of anti-HIV screens for measuring these activities can be found in: xe2x80x9cHIV Volume 2: Biochemistry, Molecular Biology and Drug Discoveryxe2x80x9d, J. Karn, ed., IRL Press, Oxford, 1995, and references therein.
6. Immunomodulatory activity:
A compendia of examples of specific screens for measuring these activities can be found in: V. St. Georgiev (1990) xe2x80x9cImmunomodulatory Activity of Small Peptidesxe2x80x9d Trends Pharm. Sci. 11, 373-378.
7. Pharmacokinetic properties:
The pharmacological activities assayed in the screening method include half-life, solubility, or stability, among others. For example, methods of analysis and measurement of pharmacokinetic properties can be found in: J.-P. Labaune xe2x80x9cHandbook of Pharmacokinetics: Toxicity Assessment of Chemicalsxe2x80x9d, Ellis Horwood Ltd., Chichester, 1989, and references therein.
In the screening method, the peptide of step a) can consist of natural amino acids. Alternatively, the peptide of step a) can contain mostly natural amino acids, but comprise one or a small number of non-natural amino acids. Such a peptide is considered to consist essentially of natural amino acids. Further in the alternative, the peptide of step a) can be mostly non-natural amino acids, but comprise one or a small number of natural amino acids. Such a peptide is considered to consist essentially of non-natural amino acids. In another alternative, the peptide of step a) can contain more than a small number and up to half non-natural amino acids or even up to mostly non-natural amino acids with the balance being natural amino acids.
In the screening method, the non-natural amino acid in the peptide of step b) can be substituted for the comparable at least one natural amino acid (e.g., lysine or arginine). The term xe2x80x9ccomparablexe2x80x9d is used herein to denote a structurally similar molecule as described in detail above. For example, the structures of the non-natural amino acids of formula I and the non-natural amino acids of formula II, III and IV are similar to and closely replicate those of the naturally occurring amino acid, lysine and arginine, respectively.
Additionally, a method of screening a peptide for an activity is also provided in which any natural amino acid in the peptide of step a) can be substituted by a non-natural amino acid having the formula I-IV in the peptide of step b). Furthermore, a screening method is provided in which the peptide of step b) can contain a non-natural amino acid of the present invention added to, rather than substituted for, a natural or non-natural amino acid in the peptide of step a).
Thus, in the screening method contemplated herein, any peptide having one or more of the present non-natural amino acids can be compared to any peptide having a known activiy to determine whether or not it has the same or similar activity at the same or different level. Depending on the specifics of how the measuring step is carried out, the present screening method can also be used to detect an activity exhibited by the peptide of step b) that differs qualitatively from the activity of the peptide of step a). Also, the screening method can be used to detect and measure differences in the same or similar activity.
Thus, present invention takes account of the situation in which the structural differences of the non-natural amino acid significantly alter the biological activity of the peptide incorporating such a non-natural amino acid. Substitution of a natural amino acid with a non-natural amino acid in a peptide typically increases the hydrophobicity of the peptide, which can result indirectly in increased binding activity when the amino acid substituted is involved in binding (e.g., receptor-ligand binding, enzyme-cofactor binding, enzyme-substrate binding) and since binding strength is correlated with activity, a peptide higher potency (higher measured activity level) can result.
Treatment
A method of treating or preventing in a subject a disease treated or prevented by the administration of a peptide comprising administering to the subject a peptide having, substituted for a natural amino acid, at least one non-natural amino acid having the formula I, II, III or IV.
As described above, the peptide used in the treatment method can be a peptide that usually contains a lysine or an arginine, but has at least one non-natural amino acid having the formula I, II, III or IV substituted therfor. Alternatively, the peptide can contain neither of these amino acids, yet it can be effective to substitute a non-natural amino acid of the invention for another amino acid. In addition the peptide used in the treatment method can have one or more non-natural amino acid of the present invention added to the existing sequence of amino acids.
In the treatment method of the present invention, the diseases that can be treated and the peptides that can be used are numerous. A partial list of peptides and diseases is set out below.
Peptides for triggering B and T cell activity can be used to treat autoimmune disease, including uveitis, collagen-induced, adjuvant and rheumatoid arthritis, thyroiditis, myasthenia gravis, multiple sclerosis and diabetes. Examples of these peptides are interleukins (referenced in Aulitzky, WE; Schuler, M; Peschel, C.; Huber, C.; Interleukins. Clinical pharmacology and therapeutic use. Drugs. 48(5):667-77, November 1994) and cytokines (referenced in Peters, M.; Actions of cytokines on the immune response and viral interactions: an overview. Hepatology. 23(4):909-16, April 1996).
Enkephlin and analogs, agonists and antagonists can be used to treat AIDS, ARC, and cancer, pain modulation, Huntington""s, Parkinson""s diseases.
LHRH and analogs, agonists and antagonists can be used to treat prostatic tumors and reproductive physiopathology, including breast cancer, and infertility.
Peptides and peptidomimetics that target crucial enzymes, oncogenes or oncogene products, tumor-suppressor genes and their products, growth factors and their corresponding receptors can be used to treat cancer. Examples of these peptides are described in Unger, C. Current concepts of treatment in medical oncology: new anticancer drugs. Journal of Cancer Research and Clinical Oncology. 122(4):189-98, 1996.
Neuropeptide Y and other pancreatic polypeptides, and analogs, agonists and antagonists can be used to treat stress, anxiety, depression and associated vasoconstrictive activities.
Gluco-incretins, including gastric inhibitory polypeptide, glucose-dependent insulinotropic polypeptide and glucagon-like polypeptide-1 and analogs, agonists and antagonists can be used to treat Type II diabetic hyperglycaemia.
Atrial natriuretic factor and analogs, agonists and antagonists can be used to treat congestive heart failure.
Integrin and analogs, agonists and antagonists can be used to treat osteoporosis, scar formation, bone synthesis, inhibition of vascular occlusion, and inhibition of tumor invasion and metastasis.
Glucagon, glucagon-like peptide 1 and analogs, agonists and antagonists can be used to treat diabetes cardiovascular emergencies.
Antithrombotic peptides and analogs, agonists and antagonists can be used to treat cardiovascular and cerebrovascular diseases. Examples of these peptides RGD, D-Phe-Pro-Arg and others named are described in Ojima I.; Chakravarty S.; Dong Q. Antithrombotic agents: from RGD to peptide mimetics. Bioorganic and Medicinal Chemistry. 3(4):337-60, 1995.
Cytokines/interleukins and analogs, agonists and antagonists can be used to treat inflammatory disease, immune response dysfunction, hematopoiesis, mycosis fungoides, aplastic anemia, thrombocytopenia, and malignant melanoma. Examples of these peptides are Interleukins, referenced in Aulitzky et al. and Peters et al.
Endothelin and analogs, agonists and antagonists can be used to treat arterial hypertension, myocardial infarction, congestive heart failure, atherosclerosis, shock conditions, renal failure, asthma and vasospasm.
Natriuretic hormones and analogs, agonists and antagonists can be used to treat cardiovasicular disease and acute renal failure. Examples of these peptides are named and described in Espiner, E. A;. Richards, A. M.; Yandle, T. G.; Nicholls, M. G.; Natriuretic hormones. Endocrinology and Metabolism Clinics of North America. 24(3):481-509, 1995.
Peptides that activate or inhibit tyrosine kinase, or bind to TK-activating or inhibiting peptides and analogs, agonists and antagonists can be used to treat chronic myelogenous and acute lymphocytic leukemias, breast and ovarian cancers and other tyrosine kinase associated diseases. Examples of these peptides are described in Smithgall, T E.; SH2 and SH3 domains: potential targets for anti-cancer drug design. Journal of Pharmacological and Toxicological Methods. 34(3):125-32, 1995.
Renin inhibitors analogs, agonists and antagonists can be used to treat cardiovascular disease, including hypertension and congestive heart failure. Examples of these peptides are described in Rosenberg, S. H.; Renin inhibition. Cardiovascular Drugs and Therapy. 9(5):645-55, 1995.
Angiotensin-converting enzyme inhibitors, analogs, agonists and antagonists can be used to treat cardiovascular disease, including hypertension and congestive heart failure.
Peptides that activate or inhibit tyrosine phosphorylases can be used to treat cardiovascular diseases. Examples of these peptides are described in Srivastava, A. K.; Protein tyrosine phosphorylation in cardiovascular system. Molecular and Cellular Biochemistry. 149-150:87-94, 1995.
Peptide based antivirals can be used to treat viral diseases. Examples of these peptides are described in Toes, R. E.; Feltkamp, M. C.; Ressing, M. E.; Vierboom, M. P.; Blom, R. J.; Brandt, R. M; Hartman, M.; Offringa, R.; Melief, C. J.; Kast, W. M.; Cellular immunity against DNA tumour viruses: possibilities for peptide-based vaccines and immune escape. Biochemical Society Transactions. 23(3):692-6, 1995.
Corticotropin releasing factor and peptide analogs, agonists and antagonists can be used to treat disease associated with high CRF, i.e Alzheimer""s disease, anorexia nervosa, depressive, psycotic disorders, arthritis, and multiple sclerosis.
Peptide agonists and antagonists of platelet-derived wound-healing formula (PDWHF) can be used as a therapy for donor tissue limitations and wound-healing constraints in surgery. Examples of these peptides are described in Rudkin, G. H.; Miller, T. A.; Growth factors in surgery. Plastic and Reconstructive Surgery. 97(2):469-76, 1996.
Fibronectin, fibribnopeptide inhibitors and analogs, agonists and antagonists can be used to treat metastasis (i.e. enzyme inhibition, tumor cell migration, invasion, and metastasis).
Chemokines (types of cytokine, including interleukin-8, RANTES, and monocyte chemotactic peptide) analogs, agonists and antagonists can be used to treat arthritis, hypersensitivity, angiogenesis, renal disease, glomerulonephritis, inflammation, and hematopoiesis.
Neutral endopeptidase inhibitors and analogs, agonists and antagonists can be used to treat hypertension and inflammation. Examples of these peptides are described in Gregoire, J. R; Sheps, S. G; Newer antihypertensive drugs. Current Opinion in Cardiology. 10(5):445-9, 1995.
Substance P and analogs, agonists and antagonists can be used to treat immune system dysfunction, pain transmission/perception and in autonomic reflexes and behaviors.
Alpha-melanocyte-stimulating hormone and analogs, agonists and antagonists can be used to treat AIDS, rheumatoid arthritis, and myocardial infarction.
Bradykinin (BK) and analogs, agonists and antagonists can be used to treat inflammatory diseases (edema, etc), asthma, allergic reactions (rhinitis, etc), anesthetic uses, and septic shock.
Secretin can be used to treat cardiovascular emergencies.
GnRH and analogs, agonists and antagonists can be used to treat hormone-dependent breast and prostate tumors.
Somatostatin and analogs, agonists and antagonists can be used to treat gut neuroendocrine tumors.
Gastrin, Gastrin Releasing Peptide and analogs, agonists and antagonists can be used as an adjuvant to chemotherapy or surgery in small cell lung cancer and other malignancies, or to treat allergic respiratory diseases, asthma and allergic rhinitis.
Laminin-derived synthetic peptides analogs, agonists and antagonists can be used to treat tumor cell growth, angiogenesis, regeneration studies, vascularization of the eye with diabetes, and ischemia. Examples of these peptides are described in Kleinman, H. K.; Weeks, B. S.; Schnaper, H. W.; Kibbey, M. C.; Yamamura, K.; Grant, D. S; The laminins: a family of basement membrane glycoproteins important in cell differentiation and tumor metastases. Vitamins and Hormones. 47:161-86, 1993.
Defensins, corticostatins, dermaseptins, mangainins, and other antibiotic (antibacterial and antimicrobial) peptides and analogs, agonists and antagonists can be used to treat infections, tissue inflammation and endocrine regulation.
Vasopressin and analogs, agonists and antagonists can be used to treat neurological disorders, stress and Diabetes insipidus.
Oxytocin and analogs, agonists and antagonists can be used to treat neurological disorders and preterm labor.
ACTH-related peptides and analogs, agonists and antagonists can be used as neurotrophic, neuroprotective, and peripheral demyelinating neuropathy agents.
Amyloid-beta peptide and analogs, agonists and antagonists can be used to treat Alzheimer""s disease.
Epidermal growth factor, receptor, and analogs, agonists and antagonists can be used to treat necrotizing enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulceration, colitis, and congenital microvillus atrophycarcinomas.
Leukocyte adhesion molecules and their ligands, and analogs, agonists and antagonists can be used to treat atherosclerosis, inflammation. Examples of these peptides are described in Barker, J. N.; Adhesion molecules in cutaneous inflammation. Ciba Foundation Symposium. 189:91-101.
Major histocompatibility complex (MHC) binding peptides and analogs, agonists and antagonists can be used to treat autoimmune, immunodysfunctional, immuno modulatory diseases and as well as used for their corresponding therapies. Examples of these peptides are described in Appella, E.; Padlan, E. A.; Hunt, D. F; Analysis of the structure of naturally processed peptides bound by class I and class II major histocompatibility complex molecules. EXS. 73:105-19, 1995.
Corticotropin releasing factor can be used to treat neurological disorders.
Neurotrophins (including brain-derived neurotrophic factor (BDNF), nerve growth factor, and neurotrophin 3) and analogs, agonists and antagonists can be used to treat neurological disorders.
Cytotoxic T-cell activating peptides can be used to treat infectious diseases and cancer. Examples of these peptides are described in: Chesnut R. W.; Sette, A.; Celis, E.; Wentworth, P.; Kubo, R. T.; Alexander, J.; Ishioka, G.; Vitiello, A.; Grey, H. M; Design and testing of peptide-based cytotoxic T-cell-mediated immunotherapeutics to treat infectious diseases and cancer. Pharmaceutical Biotechnology. 6:847-74, 1995.
Peptide immunogens for prevention of HIV-1 and HTLV-I retroviral infections can be used to treat AIDS. Examples of these peptides are described in Hart, M. K.; Palker, T. J.; Haynes, B F; Design of experimental synthetic peptide immunogens for prevention of HIV-1 and HTLV-I retroviral infections. Pharmaceutical Biotechnology. 6:821-45, 1995.
Galanin and analogs, agonists and antagonists can be used to treat Alzheimer""s disease, depression, eating disorders, chronic pain, prevention of ischemic damage, and growth hormone modulation.
Tachykinins (neurokinin A and neurokinin B) and analogs, agonists and antagonists can be used to treat pain transmission/perception and in autonomic reflexes and behaviors.
RGD containing peptides can be used to treat arious diseases involved with cell adhesion, antithrombotics, and acute renal failure.
Osteogenic growth peptide and analogs, agonists and antagonists can be used as treatment of systemic bone loss. Examples of these peptides are described in Bab IA. Regulatory role of osteogenic growth peptide in proliferation, osteogenesis, and hemopoiesis. Clinical Orthopaedics and Related Research. (313):64-8, 1995.
Parathyroid hormone, parathyroid hormone related-peptide and analogs, agonists and antagonists can be used to treat diseases affecting calcium homeostasis (hypercalcemia), bone metabolism, vascular disease, and atherosclerosis.
Kallidin and analogs, agonists and antagonists can be used to treat tissue injury or inflammation and pain signaling pathological conditions of the CNS.
T cell receptor peptide vaccines and analogs, agonists and antagonists can be used in immunotherapy. Examples of these peptides are described in Brostoff, S W; T cell receptor peptide vaccines as immunotherapy. Agents and Actionsxe2x80x94Supplements. 47:53-8, 1995.
Platelet-derived growth factor (PDGF) and analogs, agonists and antagonists can be used to treat non-neoplastic hyperproliferative disorders, therapy for donor tissue limitations and wound-healing constraints in surgery.
Amylin, calcitonin gene related peptides (CGRP) and analogs, agonists and antagonists can be used to treat insulin-dependent diabetes.
Vasoactive intestinal polypeptide and analogs, agonists and antagonists can be used to treat allergic respiratory diseases, asthma and allergic rhinitis, and nervous control of reproductive functions.
Growth hormone-releasing hormone and analogs, agonists and antagonists can be used to treat growth hormone deficiency and immunomodulation.
HIV protease inhibiting peptides can be used to treat AIDS. Examples of these peptides are described in Bugelski, P. J.; Kirsh, R.; Hart, T. K; HIV protease inhibitors: effects on viral maturation and physiologic function in macrophages. Journal of Leukocyte Biology. 56(3):374-80, 1994.
Thymopoietin active fragment peptides and analogs, agonists and antagonists can be used to treat rheumatoid arthritis and virus infections.
Cecropins and analogs, agonists and antagonists can be used as antibacterials.
Thyroid releasing hormone and analogs, agonists and antagonists can be used to treat spinal cord injury and shock.
Erythropoietin and analogs, agonists and antagonists can be used to treat anemia.
Fibroblast growth factor (FGF), receptor and analogs, agonists and antagonists can be as stimulation of bone formation, as well as used as a treatment for Kaposi""s sarcoma, neuron regeneration, prostate growth, tumor growth inhibition, and angiogenesis.
Stem cell factor and analogs, agonists and antagonists can be used to treat anemias.
GP120, GP160, CD4 fragment peptides and analogs, agonists and antagonists can be used to treat AIDS.
Insulin-like growth factor, receptor, and analogs, agonists and antagonists can be used to treat breast and other cancers, noninsulin-dependen diabetest mellitus, cell proliferation, apoptosis, hematopoiesis, AIDS, growth disorders, osteoporosis,and insulin resistance.
Colony stimulating factors (granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and macrophage colony-stimulating factor and analogs, agonists and antagonists can be used to treat anemias.
Kentsin and analogs, agonists and antagonists can be used for immunomodulation.
Lymphocyte activating peptide and analogs, agonists and antagonists can be used for immunomodulation. Examples of these peptides are described in Loleit, M.; Deres, K.; Wiesmuller, K. H.; Jung, G.; Eckert, M.; Bessler, W. G; Biological activity of the Escherichia coli lipoprotein: detection of novel lymphocyte activating peptide segments of the molecule and their conformational characterization. Biological Chemistry Hoppe-Seyler. 375(6):407-Jun. 12, 1994.
Tuftsin and analogs, agonists and antagonists can be used for immunomodulation.
Prolactin and analogs, agonists and antagonists can be used to treat rheumatic diseases, systemic lupus erythematosus, hyperprolactemia.
Angiotensin II and receptor(s) and analogs, agonists and antagonists can be used to treat hypertension, hemodynamic regulation, neurological disorders, diabetic nephropathies, aortoarterities induced RVH, hyperaldosteronism, heavy metal induced cardiovascular effects, diabetes mellitus and thyroid dysfunction.
Dynorphin and analogs, agonists and antagonists can be used to treat neurological disorders, pain management, algesia, spinal cord injury and epilepsy.
Calcitonin, and analogs, agonists and antagonists can be used to treat neurological disorders, immune system dysfunction, calcium homeostasis, and osteoporosis.
Pituitary adenylate cyclase activating polypeptide can play a role in growth, signal transduction vasoactivity roles, exact role in diseases not determined yet.
Cholecystokinin and analogs, agonists and antagonists can be used to treat feeding disorders, panic disorders, and anti-opioid properties.
Pepstatin and analogs, agonists and antagonists can be used a pepsin and HIV protease inhibitor (AIDS).
Bestatin and analogs, agonists and antagonists can be used to treat muscular dystrophy, anticancer, antileukemia, immune response modulator, and acute non-lymphocytic leukemia.
Leupeptin and analogs, agonists and antagonists can be used as a protease inhibitor, exact role in diseases not determined yet.
Luteinizing hormone and releasing hormone and analogs, agonists and antagonists can be used as a infertility male contraceptive.
Neurotensin and analogs, agonists and antagonists can be used as a antipsychotic and analgesic agent.
Motilin and analogs, agonists and antagonists can be used as for the control of gastric emptying.
Insulin and analogs, agonists and antagonists can be used to treat diabetes.
Transforming growth factor (TGF) and analogs, agonists and antagonists can be used for cell proliferation and differentiation, cancer treatment, immunoregulation, therapy for donor tissue limitations, and wound-healing constraints in surgery.
Bone morphogenetic proteins (BMPs) and analogs, agonists and antagonists can be used as therapy for donor tissue limitations, osteogenesis, and wound-healing constraints in surgery.
Bombesin and analogs, agonists and antagonists can be used to prevent the proliferation of tumor cells, modulation of feeding, and neuroendocrine functions.
Glucagon, glucagon-like peptide 1 and analogs, agonists and antagonists can be used to treat diabetes cardiovascular emergencies.
Pancreastatin, chromogranins A, B and C and analogs, agonists and antagonistsxe2x80x94conditions associated with inhibition of insulin secretion, exocrine pancreatic secretion and gastric acid secretion, and stimulation of glucagon secretion.
Endorphins and analogs, agonists and antagonists can be used to treat neurological disorders, alleviating pain, treatment of opioid abuse, obesity, and diabetes. Examples of these peptides are named and described in Dalayeun, J. F.; Nores, J. M.; Bergal, S.; Physiology of beta-endorphins. A close-up view and a review of the literature. Biomedicine and Pharmacotherapy. 47(8):311-20, 1993.
Miscellaneous opioid peptides, including (but not limited to) adrenal peptide E, alpha casein fragment, beta casomorphin, dennorphin, kyotorphin, metophamide neuropeptide FF (NPFF), melanocyte inhibiting factor, and analogues, agonists and antagonists can be used to treat neurological disorders, alleviating pain, as well as for the treatment of opioid abuse.
Vasotocin and analogues, agonists and antagonists can be used for clinical uses to be determined.
Protein kinase C and inhibitors and analogues, agonists and antagonists can be used to treat cancer, apoptosis, smooth muscle function, and Alzheimer""s disease. Examples of these peptides are named and described in Philip, P. A.; Harris, A. L; Potential for protein kinase C inhibitors in cancer therapy. Cancer Treatment and Research. 78:3-27, 1995.
Amyloid, amyloid fibrin, fragments and analogues, agonists and antagonists can be used to treat neurodegenerative diseases and diabetes.
Calpain and other calmodulin-inhibitory proteins and analogues, agonists and antagonists can be used to treat neurodegenerative disorders, cerebral ischaemia, cataracts, myocardial ischaemia, muscular dystrophy and platelet aggregation.
Charybdotoxin, Apamin and analogues, agonists and antagonists can be used for treatment of neurodegenerative diseases and pain and cerebral ischemia.
Phospholipase A2 and receptor inhibiting/activating peptides and analogues, agonists and antagonists can be used to treat acute pancreatitis, pancreatic cancer, abdominal trauma, and inflammation, e.g., sepsis, infections, acute pancreatitis, various forms of arthritis, cancer, complications of pregnancy, and postoperative states.
Potassium channel activating and inhibiting proteins and analogues, agonists and antagonists can be used to treat various diseases. Examples of these peptides are described in Edwards, G.; Weston, A. H; Pharmacology of the potassium channel openers. Cardiovascular Drugs and Therapy. 9 Suppl 2:185-93, March 1995
IgG activators, inhibitors and analogues, agonists and antagonists can be used to treat autoimmune diseases and immune dysfunctions. Examples of these peptides are described in Mouthon, L.; Kaveri, S. V.; Spalter, S. H.; Lacroix-Desmazes, S.; Lefranc, C.; Desai, R.; Kazatchkine, M. D; Mechanisms of action of intravenous immune globulin in immune-mediated diseases. Clinical and Experimental Immunology. 104 Suppl 1:3-9, 1996.
Endotoxin and inhibitors and analogues, agonists and antagonists can be used for decreasing cardiac output, systemic hypotension, decreased blood flow and O2 delivery to tissues, intense pulmonary vasoconstriction and hypertension, bronchoconstriction, increased permeability, pulmonary oedema, ventilation-to-perfusion inequalities, hypoxaemia, and haemoconcentration. Examples of these peptides are named and described in Burrell, R; Human responses to bacterial endotoxin. Circulatory Shock. 43(3):137-53, July 1994.
Experimental
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in xc2x0 C. and is at room temperature, and pressure is at or near atmospheric.